The Norwalk and Norwalk-like viruses in the Caliciviridae are major etiologic agents of of epidemic gastroenteritis in humans. The inability to grow these human caliciviruses in cell culture makes it impossible to directly analyze either viral interactions with cellular receptors or replication in host cells. The model system for analyzing the replication of caliciviruses that we have developed in our laboratory is the growth of the feline calicivirus (FCV) in cell culture, because this virus is one of the few cultivatable representatives of this family. The infectivity of capped RNA molecules synthesized in vitro from plasmid DNA containing a full-length cDNA copy of the virus genome has been studied in detail in order to: (1) develop and optimize a recovery system and (2) identify the function of different regions of the virus genome involved in its replication. This year, we demonstrated the recovery of infectious FCV virions following simultaneous transfection of cells with plasmid DNA containing a full-length copy of the FCV genome downstream of the T7 promoter and infection of these cells by vaccinia virus (strain MVA/T7, provided by Dr. B. Moss, NIAID) expressing the gene encoding T7 RNA polymerase. This strategy allowed us to recover infectious FCV virions from cells that are not normally permissive for FCV infection. At the same time, the direct use of plasmid DNA simplified the procedure for examining the viability of genetically engineered virus mutants. Progress was made in the mapping of the nonstructural genes encoded in the ORF1 polyprotein of the FCV genome and in the analysis of its proteolytic processing. Boundaries of the core sequence of the viral 3C-like protease participating in the processing of the polyprotein and in the cleavage of the precursor of the capsid protein were determined. The site-specificity of this protease was determined by mutational analysis of the cleavage site.